Cystic fibrosis (CF) is the most predominant lethal autosomal recessive genetic disorder in Caucasians, with affected individuals occurring in approximately 1/3,000 live births; incidence is lower in other ethnic groups (Heim, et al., Genetics in Medicine 3(3):168-176 (2001)). CF disease is associated with high morbidity and reduced life span. Individuals carrying two defective CF chromosomes typically display a panoply of symptoms, including sinopulmonary disease, pancreatic insufficiency, and male infertility. Certain bacterial infections, e.g. Pseudomonas aeruginosa, are typically found only in individuals affected by CF (Raman, et al., Pediatrics 109(1): E13 (2002)). CFTR mutations are implicated in a broad spectrum of diseases such as congenital bilateral absence of the vas deference (CBAVD) (Dumur, et al., Hum Genet 97: 7-10 (1996)), allergic bronchopulmonary aspergillosis, and isolated chronic pancreatitis (Raman, supra). Moreover, disease manifestations may be exacerbated in some cases by additional environmental risk factors such as smoking, alcohol consumption, or allergy (Raman, supra).
Approximately one in 25 to 30 Caucasians is a CF carrier (Grody, Cutting, et al., Genetics in Medicine 3(2):149-154 (2001)); however, no noticeable defects or biochemical or physiological alterations can be readily used to ascertain carrier status (Grody and Desnick, Genetics in Medicine 3(2):87-90 (2001)). Determination of carrier status, as well as confirmation of CF disease, may be of value in genetic counseling as well as in early diagnosis to determine treatment and disease management (Grody and Desnick, supra). There is currently no cure for the disease, although recent advances in palliative treatments have dramatically improved the quality of life and overall longevity of affected individuals.
Diagnosis of CF has been accomplished using various means since the 1950's and often requires positive results obtained using more than one clinical parameter (Rosenstein and Cutting, Journal of Pediatrics 132(4): 589-595 (1998)). In some cases, definitive diagnosis can remain elusive for years (Rosenstein and Cutting, supra). Sweat chloride testing, involving measurement of chloride in sweat following iontophoresis of pilocarpineis a widely used procedure, although there are reports of CF affected individuals with normal sweat chloride levels, even upon repeat testing (LeGrys, Laboratory Medicine 33(1): 55-57 (2002)). Nasal potential difference, involving bioelectrical measurements of the nasal epithelium, is another clinical method that has been used to detect CF in individuals with normal sweat chloride levels (Wilson, et al., Journal of Pediatrics 132 (4): 596-599 (1998)). Immunoreactive trypsinogen (IRT) levels have been used alone as well as in combination with mutational analysis for neonatal analysis (Gregg, et al., Pediatrics 99(6): 819-824 (1997)). Elevated IRT levels are suggestive of CF disease, although the IRT assay alone has low positive predictive value, often requires repeat testing (Gregg, et al., supra), and is complicated by age-related declines in IRT values beyond 30 days (Rock, et al., Pediatrics 85(6): 1001-1007 (1990)).
The CFTR gene was first identified in 1989. The gene is located on chromosome 7, includes 27 exons, and spans 250kb (Kerem, et al., Science 245: 1073-1080 (1989); Riordan, et al., Science 245: 1066-1073 (1989); Rommens, et al., Science 245: 1059-1065 (1989)). CFTR encodes a chloride ion channel; defect-causing lesions in the gene result in abnormal intracellular chloride levels, leading to thickened mucosal secretions, which in turn affect multiple organ systems. More than 950 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (ref CFGAC). One mutation, ΔF508, causes the loss of a phenylalanine residue at amino acid 508 in CFTR gene product and accounts for 66% of defective CF chromosomes worldwide (Bobadilla, et al., Human Mutation 19: 575-606 (2002)). The remaining alleles exhibit considerable ethnic and regional heterogeneity (Bobadilla, et al., supra) and, in many cases, exhibit poor genotype-phenotype correlations (Grody, Cutting et al., supra). Severity of CF disease in individuals affected by more rare mutations is highly variable. In some cases, a typical, moderate, or partial CF disease may be the result of a partially functional CFTR gene product (Noone and Knowles, Respiratory Research 2(6):328-332 (2001)).
The identification of the CFTR gene enabled significant advances in CF diagnosis and carrier screening. However, use of genetics to establish carrier status or the presence of CF disease remains challenging for several reasons. First, the number of exons and the overall size of the CFTR gene complicate analysis. Most methods applied to CF testing rely on PCR to amplify the more than 15 different exons and intronic regions found thus far to contain the most frequently encountered mutations; the amplicons are then tested individually to determine which mutations, if any, are present. Second, the number of mutations identified in the CFTR gene has increased steadily. As recently as 1994, 400 mutations had been identified; that number grew to more than 950 by 2002 ((Cystic Fibrosis Genetic Analysis Consortium (CFGAC) 2002) and is likely to continue to increase. The existence of so many distinct alleles complicates the use of a number of standard mutation detection methods such as PCR-RFLP or AS-PCR. Third, many of rarely encountered alleles appear to exhibit incomplete penetrance (Grody, Cutting et al. supra) and may be associated with heterologous genetic alterations (Raman, et al., supra; Rohlfs, et al., Genetics in Medicine 4(5):319-323 (2002)). Fourth, some alleles, such as R117H, produce different phenotypes depending on chromosomal background (Kiesewetter, et al., Nature Genetics 5(3): 274-278 (1993)). Despite these challenges, widespread genetic screening for CF has been recommended for Caucasian and Ashkenazi Jewish couples and made available to other ethnic groups in the U.S. considering pregnancy or already expecting (Grody, Cutting et al. supra). The American College of Obstetrics and Gynecology (ACOG), the American College of Medical Genetics (AMCG), and the National Center for Human Genomics Research (NCHGR) of the NIH have together agreed upon an initial panel of 25 mutations commonly found in North America, including (F508, to be used for prenatal and carrier screening in the US (Grody, Cutting et al. supra). This panel is more inclusive for mutations affecting certain ethnic groups than some others, particularly Ashkenazi Jews and Caucasians of North European, non-Jewish descent. Nonetheless, the joint committee concluded that all couples seeking to have a child could benefit from screening that would identify, at a minimum, 50-65% of CFTR mutations. Future recommendations will likely expand the core collection of alleles to be screened in order to encompass a greater percentage of the alleles found in other sub-populations.
The case of the most commonly encountered CF allele, ΔF508, presents a particular challenge to nucleic acid-based detection methods. This region contains three polymorphisms that do not cause CF but may interfere with hybridization of wild type probes (Grody, Cutting et al. 2001). These variations result in the following amino acid changes: F508C, I507V and I506V. This situation is complicated by the existence of the CF-causing mutation ΔI507. Many methods applied to CF genotyping rely on the use of reflex tests to distinguish these benign polymorphisms from the CF-causing mutations in codons 507 and 508. Assays that rely primarily on the stringency of annealing of an oligonucleotide to a target sequence, e.g. PCR, SBH can yield false positive or negative results in the presence of such polymorphisms (Fujimura, Northrup et al. 1990).
What is needed are detection assays that may be applied directly to the analysis of CTFR sequences (e.g. genomic sequences), as well as assays capable of detecting multiple CTFR alleles in a single reaction vessel.